This exploratory R21 application proposes studies to identify and characterize the mechanism by which the vacuolating cytotoxin (VacA), an important virulence factor produced by the human gastric pathogen Helicobacter pylori, targets mitochondria within intoxicated host cells. VacA is a membrane-channel forming toxin, which subsequent to binding the surface of host cells, is taken up from the plasma membrane into VacA- containing vesicles (VCVs), and subsequently targets the inner membrane of mitochondria, resulting in metabolic dysfunction and cell death. Here, we propose studies that challenge canonical models of host cell intoxication by intracellular-acting bacterial toxins, whic generally require the translocation of enzymatic active fragments from the endolysosomal system or endoplasmic reticulum to the cytosol prior to toxin- mediated modification of intracellular targets. Rather, subsequent to internalization, we propose that VacA is transported within VCVs to mitochondria, where the toxin is then directly transferred to mitochondria. However, the identity of VCV proteins important for VacA targeting/trafficking to mitochondria is difficult to predict, due in part to the fact that until very recently, mitochondria were not generlly thought to receive protein cargo from the endolysosomal system and, as a consequence, almost nothing is known about intracellular trafficking compartments destined for mitochondria. To address this gap in knowledge, we will evaluate the hypothesis that VacA-targeting of mitochondria requires remodeling of VacA-containing vesicles (VCVs) from early endosomal-like compartments to vesicles that are functionally competent for targeting and fusion with mitochondria. Using magnetic-based separation methods to isolate iron-enriched VCVs from cells at different stages of intoxication for proteome analysis, we will identify changes in VCV-associated proteins, and, characterize their functional importance for VacA targeting of mitochondria and mitochondrial dysfunction. These studies will provide the first detailed characterization of the host cell machinery that is important for mitochondrial targeting, and, contribute to our understanding of the fundamental biology underlying the modulation of host cells by VacA.